Inactivated staphylococcal whole-cell vaccine

ABSTRACT

A vaccine is disclosed that is protective against pathogenic bacterial species, typically staphylococcal species, and includes methods to prepare said vaccine and to culture pathogenic bacteria.

The invention relates to a vaccine that is protective against pathogenicbacterial species, typically staphylococcal species, and includingmethods to prepare said vaccine and to culture pathogenic bacteria.

Vaccines protect against a wide variety of infectious diseases. Manyvaccines are produced by inactivated or attenuated pathogens which areinjected into a subject. The immunised subject responds by producingboth a humoral (e.g. antibody) and cellular (e.g. cytolytic T cells)responses For example, some influenza vaccines are made by inactivatingthe virus by chemical treatment with formaldehyde, likewise the Salkpolio vaccine comprises whole virus inactivated with propionolactone.For many pathogens chemical or heat inactivation while it may give riseto vaccine immunogens that confer protective immunity also gives rise toside effects such as fever and injection site reactions. In the case ofbacteria, inactivated organisms tend to be so toxic that side effectshave limited the application of such crude vaccine immunogens (e.g. thecellular pertussis vaccine) and therefore vaccine development has laggedbehind drug-development. This is unfortunate as current antibiotictreatments are now prejudiced by the emergence of drug-resistantbacteria.

An example of a pathogenic organism which has developed resistance toantibiotics is Staphylococcus aureus. S. aureus is a bacterium whosenormal habitat is the epithelial lining of the nose in about 20-40% ofnormal healthy people and is also commonly found on people's skinusually without causing harm. However, in certain circumstances,particularly when skin is damaged, this pathogen can cause infection.This is a particular problem in hospitals where patients may havesurgical procedures and/or be taking immunosuppressive drugs. Thesepatients are much more vulnerable to infection with S. aureus because ofthe treatment they have received. Resistant strains of S. aureus havearisen in recent years. Methicillin resistant strains are prevalent andmany of these resistant strains are also resistant to several otherantibiotics. Currently there is no effective vaccination procedure forS. aureus.

S. aureus is therefore a major human pathogen capable of causing a widerange of diseases some of which are life threatening diseases includingsepticaemia, endocarditis, arthritis and toxic shock. This ability isdetermined by the versatility of the organism and its arsenal ofcomponents involved in virulence. At the onset of infection, and as itprogresses, the needs and environment of the organism changes and thisis mirrored by a corresponding alteration in the virulence determinantswhich S. aureus produces. At the beginning of infection it is importantfor the pathogen to adhere to host tissues and so a large repertoire ofcell surface associated attachment proteins are made. These includecollagen-, fibrinogen- and fibronectin-binding proteins. The pathogenalso has the ability to evade host defences by the production of factorsthat reduce phagocytosis or interfere with the ability of the cells tobe recognised by circulating antibodies. Often a focus of infectiondevelops as an abscess and the number of organisms increases. S. aureushas the ability to monitor its own cell density by the production of aquorum sensing peptide. Accumulation of the peptide, associated withphysiological changes brought about by the beginning of starvation ofthe cells, elicits a switch in virulence determinant production fromadhesins to components involved in invasion and tissue penetration.These include a wide range of hemolysins, proteases and otherdegradative enzymes; (see also, Manual of Clinical Microbiology FourthEdition. Editors Edwin H Lennette, Albert Balows, William J Hausler Jr,H Jean Shadomy; published by the Americal Society for Microbiology1985).

We disclose the development of a chloroform-inactivated whole bacterialvaccine using a clinical isolate of S. aureus which is selected as beingrepresentative of a spectrum of S. aureus strains which were tested by anumber of criteria. The antigenicity of the vaccine designated SA75 hasbeen demonstrated by enzyme linked immunosorbent assay (ELISA) andwestern blotting with hyperimmune rabbit serum and the vaccine produceda dose related immune response in both male and female rabbits.Preliminary data from a placebo controlled double blind Phase I clinicaltrial of the vaccine in human male volunteers demonstrated that thevaccine was both safe and immunogenic. It also cross-reacts with manyother pathogenic bacteria and therefore provides a vaccine that isprotective to a wide range of bacterial pathogens. In addition wedescribe cell culture conditions that are substantially free of animalderived products and the use of these conditions in vaccine production.

According to an aspect of the invention there is provided a vaccinecomposition comprising an inactivated staphylococcal cell wherein saidcomposition is prepared using a staphylococcal cell characterised inthat said cell:

-   -   i) is a gram positive cocci;    -   ii) expresses at least the enzyme catalase;    -   iii) induces an immune response that produces antibodies that        bind at least staphylococcal collagen-binding protein; and    -   iv) is resistant to the antibiotic penicillin.

The staphylococcal bacterial cell is characterised by a number ofbiological and biochemical features that include the expression ofselected genes (e.g., urease and arginine dihydrolase); the sensitivityto a number of antibiotics and including the ability to metabolisecarbohydrate sources, the reduction of nitrate and methyl carbinol

In a preferred embodiment of the invention said staphylococcal cell alsoexpresses the enzymes coagulase and/or Dnase.

In a further preferred embodiment of the invention said staphylococcalcell induces an immune response that produces antibodies that bindcollagen binding protein.

In a further preferred embodiment of the invention said inactivatedstaphylococcal cell induces an immune response that produces antibodiesthat cross react with methicillin resistant, vancomycin resistant andvancomycin intermediate resistant staphylococcal species.

In a further preferred embodiment of the invention said staphylococcalcell is sensitive to the antibiotics cloxacillin, erythromycin,tetracycline and gentamicin.

In a preferred embodiment of the invention said staphylococcal cell isselected from the group consisting of: S. epidermidis, S. aureus, S.hominis, S. haemolyticus, S. warneri, S. capitis, S. saccharolyticus, S.auricularis, S. simulans, S. saprophyticus, S. cohnii, S. xylosus, S.cohnii, S. warneri, S. hyicus, S. caprae, S. gallinarum, S. intermedius,S. hominis.

In a further preferred embodiment of the invention said staphylococcalcell is S. aureus or S. epidermidis.

In a further preferred embodiment of the invention said staphylococcalcell is an antibiotic resistant staphylococcal cell.

In a preferred embodiment of the invention said antibiotic resistancestaphylococcal cell is a methicillin resistant staphylococcal cell(MRSA).

In an alternative preferred embodiment of the invention said antibioticresistance staphylococcal cell is a vancomycin resistant staphylococcalcell (VRSA).

In a further preferred embodiment of the invention said staphylococcalcell is a Staphylococcus aureus cell designated as P/DFO 75 (NationalCollection of Type Cultures (NCTC) deposited on 19^(th), June 2007;accession number 13408; Deposited under the Budapest Treaty on theInternational Recognition of the deposit of Micro-organisms as amendedin 1980).

In a preferred embodiment of the invention said staphylococcal cell isprovided at a protein concentration of not more than about 1 mgbacterial protein/ml.

In a preferred embodiment of the invention said staphylococcal cell isprovided at a protein concentration of not more than about 0.45 mgbacterial protein/ml.

In a further preferred embodiment of the invention said staphylococcalcell is provided at a protein concentration of at least 0.0001 mgbacterial protein/ml

In a further preferred embodiment of the invention said staphylococcalcell is provided at a protein concentration of at least 0.1 mg bacterialprotein/ml

In a further preferred embodiment of the invention said staphylococcalcell is provided at a protein concentration of between 0.0001-1 mgbacterial protein/ml.

In a further preferred embodiment of the invention said staphylococcalcell is provided at a protein concentration of between 0.1-0.45 mgbacterial protein/ml.

In a still further preferred embodiment of the invention saidstaphylococcal cell is provided at between 0.25-0.36 mg bacterialprotein/ml.

In a further preferred embodiment of the invention said staphylococcalcell is provided at about 0.35 mg bacterial protein/ml.

In a preferred embodiment of the invention said vaccine compositioncomprises an adjuvant and/or excipient.

An adjuvant is a substance or procedure which augments specific immuneresponses to antigens by modulating the activity of immune cells.Examples of adjuvants include, by example only, agonistic antibodies toco-stimulatory molecules, Freunds adjuvant, muramyl dipeptides,liposomes. An adjuvant is therefore an immunomodulator.

The vaccine compositions of the invention can be administered by anyconventional route, including injection, intranasal spray by inhalationof for example an aerosol or nasal drops, or by gradual infusion overtime The administration may be, for example, intravenous,intraperitoneal, intramuscular, intracavity, subcutaneous, ortransdermal. The vaccine compositions of the invention are administeredin effective amounts. An “effective amount” is that amount of a vaccinecomposition that alone or together with further doses, produces thedesired response. In the case of treating a particular bacterial diseasethe desired response is providing protection when challenged by aninfective agent.

In a preferred embodiment of the invention said vaccine composition isadapted for administration as a nasal spray.

In a preferred embodiment of the invention said vaccine composition isprovided in an inhaler and delivered as an aerosol.

According to a further aspect of the invention there is provided aninhaler comprising a vaccine composition according to the invention.

Such amounts of vaccine will depend, of course, on the individualpatient parameters including age, physical condition, size and weight,the duration of the treatment, the nature of concurrent therapy (ifany), the specific route of administration and like factors within theknowledge and expertise of the health practitioner. These factors arewell known to those of ordinary skill in the art and can be addressedwith no more than routine experimentation.

It is generally preferred that a maximum dose of the individualcomponents or combinations thereof be used sufficient to provokeimmunity; that is, the highest safe dose according to sound medicaljudgment. It will be understood by those of ordinary skill in the art,however, that a patient may insist upon a lower dose or tolerable dosefor medical reasons, psychological reasons or for virtually any otherreasons.

The vaccine compositions used in the foregoing methods preferably aresterile and contain an effective amount of staphylococci for producingthe desired response in a unit of weight or volume suitable foradministration to a patient. The doses of vaccine administered to asubject can be chosen in accordance with different parameters, inparticular in accordance with the mode of administration used and thestate of the subject. Other factors include the desired period oftreatment. In the event that a response in a subject is insufficient atthe initial doses applied, higher doses (or effectively higher doses bya different, more localized delivery route) may be employed to theextent that patient tolerance permits.

In general, doses of vaccine are formulated and administered in dosesbetween 0.1 mg and 0.45 mg and preferably between 0.15 mg and 0.4 mg,according to any standard procedure in the art. Other protocols for theadministration of the vaccine compositions will be known to one ofordinary skill in the art, in which the dose amount, schedule ofinjections, sites of injections, mode of administration and the likevary from the foregoing. Administration of the vaccine compositions tomammals other than humans, (e.g. for testing purposes or veterinarytherapeutic purposes), is carried out under substantially the sameconditions as described above. A subject, as used herein, is a mammal,preferably a human, and including a non-human primate, cow, horse, pig,sheep, goat, dog, cat or rodent.

When administered, the vaccine compositions of the invention are appliedin therapeutically acceptable amounts and in therapeutically acceptablecompositions. The term “therapeutically acceptable” means a non-toxicmaterial that does not interfere with the effectiveness of thebiological activity of the active ingredients. Such preparations mayroutinely contain salts, buffering agents, preservatives, compatiblecarriers, and optionally other anti-bacterial agents. The vaccinecompositions may contain suitable buffering agents, including: aceticacid in a salt; citric acid in a salt; boric acid in a salt; andphosphoric acid in a salt.

The vaccine compositions also may contain, optionally, suitablepreservatives, such as: benzalkonium chloride; chlorobutanol; parabensand thiomerosal

Vaccine compositions suitable for parenteral administration convenientlycomprise a sterile aqueous or non-aqueous preparation of vaccine, whichis preferably isotonic with the blood of the recipient. This vaccine maybe formulated according to known methods using suitable dispersing orwetting agents and suspending agents. The sterile injectable preparationalso may be a sterile injectable solution or suspension in a non-toxicparenterally-acceptable diluent or solvent, for example, as a solutionin 1,3-butane diol. Among the acceptable vehicles and solvents that maybe employed are water, Ringer's solution, and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose any bland fixed oilmay be employed including synthetic mono-or di-glycerides. In addition,fatty acids such as oleic acid may be used in the preparation ofinjectables. Carrier formulation suitable for subcutaneous, intravenous,intramuscular, etc. administrations can be found in Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa.

In a preferred embodiment of the invention there is provided a vaccinecomposition according to the invention that includes at least oneadditional anti-bacterial agent.

In a preferred embodiment of the invention said agent is a seconddifferent vaccine and/or immunogenic agent (for example a bacterialpolypeptide and/or polysaccharide antigen).

According to a further aspect of the invention there is provided astaphylococcal cell characterised in that said cell:

-   -   i) is a gram positive cocci;    -   ii) expresses at least the enzyme catalase;    -   iii) induces an immune response that produces antibodies that        bind at least staphylococcal collagen-binding protein;    -   iv) is resistant to the antibiotic penicillin;        for use in the manufacture of a vaccine composition for the        vaccination of an animal subject with respect to a bacterial        infection wherein said infection is not caused by a        staphylococcal bacterial cell.

In a preferred embodiment of the invention said bacterial infection iscaused by at least one bacterial cell selected from the group consistingof: Enterococcus faecalis; Mycobacterium tuberculsis; Streptococcusgroup B; Streptoccocus pneumoniae; Helicobacter pylori; Neisseriagonorrhoea; Streptococcus group A; Borrelia burgdorferi; Coccidiodesimmitis; Histoplasma sapsulatum; Klebsiella edwardii; Neisseriameningitidis type B; Proteus mirabilis; Shigella flexneri; Escherichiacoli; Haemophilus influenzae, Chalmydia trachomatis, Chlamydiapneumoniae, Chlamydia psittaci, Francisella tularensis, Pseudomonasaeruginos, Bacillus anthracis, Clostridium botulinum, Yersinia pestis,Burkholderia mallei or B pseudomallei.

According to a further aspect of the invention there is provided astaphylococcal cell characterised in that said cell:

-   -   i) is a gram positive cocci;    -   ii) expresses at least the enzyme catalase;    -   iii) induces an immune response that produces antibodies that        bind at least staphylococcal collagen-binding protein;    -   iv) is resistant to the antibiotic penicillin;        for use in the manufacture of a vaccine composition for the        vaccination of an animal subject with respect to a yeast        infection.

In a preferred embodiment of the invention said yeast infection iscaused by a pathogenic yeast species, for example Candida albicans orSaccharomyces cerevisiae.

In a preferred embodiment of the invention said yeast infection isassociated with an immune suppressed state; for example an immunesuppressed state as a consequence of an HIV infection or as a result ofthe administration of immunosuppressive drugs.

In a preferred embodiment of the invention said animal subject is human.

According to a further aspect of the invention there is provided amethod to vaccinate an animal against a bacterial infection comprisingadministering an effective amount of the vaccine composition accordingto the invention.

In a preferred method of the invention said animal is a human.

In a preferred method of the invention said bacterial infection iscaused by a bacterial pathogen selected from the group consisting of:Enterococcus faecalis; Mycobacterium tuberculsis; Streptococcus group B;Streptoccocus pneumoniae; Helicobacter pylori; Neisseria gonorrhoea;Streptococcus group A; Borrelia burgdorferi; Coccidiodes immitis;Histoplasma sapsulatum; Klebsiella edwardii; Neisseria meningitidis typeB; Proteus mirabilis; Shigella flexneri; Escherichia coli; Haemophilusinfluenzae, Chalmydia trachomatis, Chlamydia pneumoniae, Chlamydiapsittaci, Francisella tularensis, Pseudomonas aeruginos, Bacillusanthracis, Clostridium botulinum, Yersinia pestis, Burkholderia malleior B pseudomallei.

In a preferred method of the invention said bacterial infection iscaused by a bacterial cell selected from the group consisting of: S.epidermidis, S. aureus, S. hominis, S. haemolyticus, S. warneri, S.capitis, S. saccharolyticus, S. auricularis, S. simulans, S.saprophyticus, S. cohnii, S. xylosus, S. cohnii, S. warneri, S. hyicus,S. caprae, S. gallinarum, S. intermedius, S. hominis.

In a further preferred method of the invention said bacterial species isS. aureus or S. epidermidis.

In a further preferred method of the invention said bacterial infectionis caused by an antibiotic resistant bacterial cell; preferably astaphylococcal bacterial cell.

In a preferred method of the invention said antibiotic resistancestaphylococcal cell is a methicillin resistant staphylococcal species(MRSA).

In an alternative preferred method of the invention said antibioticresistance staphylococcal cell is a vancomycin resistant staphylococcalcell (VRSA).

A preferred route of administration is intradermal, subcutaneous,intramuscular or intranasal (e.g. as an aerosol); however thevaccination method is not restricted to a particular mode ofadministration.

In a preferred method of the invention said bacterial infection resultsin a disease associated with a staphylococcal infection.

A staphylococcal associated disorder may include , for example,tuberculosis; bacteria-associated food poisoning; blood infections;peritonitis; endocarditis; osteomyelitis; sepsis; skin disorders,meningitis; pneumonia; stomach ulcers; gonorrhoea; strep throat;streptococcal-associated toxic shock; necrotizing fasciitis; impetigo;histoplasmosis; Lyme disease; gastro-enteritis; dysentery; shigellosis;and arthritis.

In an alternative preferred method of the invention said animal is alive stock animal.

In a preferred method of the invention said live stock animal isvaccinated against bacterial mastitis caused by gram positive cocci;preferably staphylococcal and/or streptococcal bacterial cells.

In a preferred method of the invention said bacterial mastitis is causedby Staphylococcus aureus and/or Streptococcus agalactiae.

In a preferred method of the invention said life stock animal is acaprine animal (e.g. sheep, goat).

In a preferred method of the invention said life stock animal is abovine animal (e.g. a cow).

Staphylococcal mastitis is a serious condition that affects live stockand can result in considerable expense with respect to controlling thedisease through administration of antibiotics and in terms of lost milkyield. The vaccine according to the invention provides cost effectivecontrol of bacterial, in particular staphylococcal mastitis.

According to a further aspect of the invention there is provided amethod for preparing a hybridoma cell-line producing monoclonalantibodies that bind staphylococcal bacterial polypeptides comprisingthe steps of:

-   -   i) vaccinating an immunocompetent mammal with a vaccine        composition according to the invention;    -   ii) fusing lymphocytes of the vaccinated immunocompetent mammal        with myeloma cells to form hybridoma cells;    -   iii) screening monoclonal antibodies produced by the hybridoma        cells of step (ii) for binding activity with respect to        staphylococcal bacterial polypeptides;    -   iii) cloning the hybridoma cells and culturing the cells to        proliferate and to secrete said monoclonal antibody; and    -   iv) recovering the monoclonal antibody from the culture        supernatant.

Preferably, said immunocompetent mammal is a mouse. Alternatively, saidimmunocompetent mammal is a rat.

The production of monoclonal antibodies using hybridoma cells iswell-known in the art. The methods used to produce monoclonal antibodiesare disclosed by Kohler and Milstein in Nature 256, 495-497 (1975) andalso by Donillard and Hoffman, “Basic Facts about Hybridomas” inCompendium of Immunology V.II ed. by Schwartz, 1981, which areincorporated by reference.

According to an aspect of the invention there is provided a hybridomacell line formed by the method according to the invention.

According to a yet further aspect of the invention there is provided amonoclonal antibody produced by the hybridoma cell-line according to theinvention.

In a preferred embodiment of the invention said monoclonal antibody isan opsonic antibody.

Phagocytosis is mediated by macrophages and polymorphic leukocytes andinvolves the ingestion and digestion of micro-organisms, damaged or deadcells, cell debris, insoluble particles and activated clotting factors.Opsonins are agents which facilitate the phagocytosis of the aboveforeign bodies. Opsonic antibodies are therefore antibodies whichprovide the same function. Examples of opsonins are the Fc portion of anantibody or compliment C3.

In a further preferred embodiment of the invention said monoclonalantibody or preferably opsonic antibody is chimeric or humanized byrecombinant techniques to combine the complimentarity determiningregions of said antibody with both the constant (C) regions and theframework regions from the variable (V) regions of a human antibody.

Chimeric antibodies are recombinant antibodies in which all of theV-regions of a mouse or rat antibody are combined with human antibodyC-regions. Humanised antibodies are recombinant hybrid antibodies whichfuse the complimentarity determining regions from a rodent antibodyV-region with the framework regions from the human antibody V-regions.The C-regions from the human antibody are also used. The complimentaritydetermining regions (CDRs) are the regions within the N-terminal domainof both the heavy and light chain of the antibody to where the majorityof the variation of the V-region is restricted. These regions form loopsat the surface of the antibody molecule. These loops provide the bindingsurface between the antibody and antigen. Antibodies from non-humananimals provoke an immune response to the foreign antibody and itsremoval from the circulation. Both chimeric and humanised antibodieshave reduced antigenicity when injected to a human subject because thereis a reduced amount of rodent (i.e. foreign) antibody within therecombinant hybrid antibody, while the human antibody regions do notillicit an immune response. This results in a weaker immune response anda decrease in the clearance of the antibody.

In a further preferred embodiment of the invention there is provided anactive binding fragment of said monoclonal antibody.

Various fragments of antibodies are known in the art, i.e., Fab, Fab₂,F(ab′)₂, Fv, Fc, Fd, scFvs, etc. A Fab fragment is a multimeric proteinconsisting of the immunologically active portions of an immunoglobulinheavy chain variable region and an immunoglobulin light chain variableregion, covalently coupled together and capable of specifically bindingto an antigen. Fab fragments are generated via proteolytic cleavage(with, for example, papain) of an intact immunoglobulin molecule. A Fab₂fragment comprises two joined Fab fragments. When these two fragmentsare joined by the immunoglobulin hinge region, a F(ab′)₂ fragmentresults. An Fv fragment is multimeric protein consisting of theimmunologically active portions of an immunoglobulin heavy chainvariable region and an immunoglobulin light chain variable regioncovalently coupled together and capable of specifically binding to anantigen. A fragment could also be a single chain polypeptide containingonly one light chain variable region, or a fragment thereof thatcontains the three CDRs of the light chain variable region, without anassociated heavy chain variable region, or a fragment thereof containingthe three CDRs of the heavy chain variable region, without an associatedlight chain moiety; and multi specific antibodies formed from antibodyfragments, this has for example been described in U.S. Pat. No.6,248,516. Fv fragments or single region (domain) fragments aretypically generated by expression in host cell lines of the relevantidentified regions. These and other immunoglobulin or antibody fragmentsare within the scope of the invention and are described in standardimmunology textbooks such as Paul, Fundamental Immunology or Janeway etal. Immunobiology (cited above). Molecular biology now allows directsynthesis (via expression in cells or chemically) of these fragments, aswell as synthesis of combinations thereof. A fragment of an antibody canalso have bispecific function as described above.

In a preferred embodiment of the invention there is provided human seraobtained by vaccination of a human subject with a vaccine compositionaccording to the invention.

In a preferred embodiment of the invention there is provided a humanantibody obtained by vaccination of a human subject with a vaccinecomposition according to the invention.

In a preferred embodiment of the invention said human antibody is anisotype selected from the group consisting of: IgA, IgM, IgD, IgE andIgG.

According to a further aspect of the invention there is provide the useof human sera obtained by vaccination with a vaccine compositionaccording to the invention in the manufacture of a medicament for thetreatment of a bacterial infection.

In a preferred embodiment of the invention said bacterial infection is astaphylococcal infection.

According to a further aspect of the invention there is provide the useof a human antibody obtained by vaccination with a vaccine compositionaccording to the invention in the manufacture of a medicament for thetreatment of a bacterial infection.

In a preferred embodiment of the invention said bacterial infection is astaphylococcal infection.

According to a further aspect of the invention there is provided amethod to prepare a vaccine to a bacterial pathogen comprising the stepsof:

-   -   i) forming a cell culture preparation comprising at least one        bacterial pathogen and nutrient broth comprising plant derived        products;    -   ii) culturing said cell culture preparation; and    -   iii) contacting said cell culture preparation with an agent that        inactivates said bacterial pathogen.

In a preferred method of the invention said bacterial pathogen isselected from the group consisting of: Staphylococcus aureus;Staphylococcus epidermidis; Enterococcus faecalis; Mycobacteriumtuberculsis; Streptococcus group B; Streptoccocus pneumoniae;Helicobacter pylori; Neisseria gonorrhoea; Streptococcus group A;Borrelia burgdorferi; Coccidiodes immitis; Histoplasma sapsulatum;Klebsiella edwardii; Neisseria meningitidis type B; Proteus mirabilis;Shigella flexneri; Escherichia coli; Haemophilus influenzae, Chalmydiatrachomatis, Chlamydia pneumoniae, Chlamydia psittaci, Francisellatularensis, Pseudomonas aeruginos, Bacillus anthracis, Clostridiumbotulinum, Yersinia pestis, Burkholderia mallei or B pseudomallei.

In a preferred method of the invention said bacterial pathogen isselected from the group consisting of: S. epidermidis, S. aureus, S.hominis, S. haemolyticus, S. warneri, S. capitis, S. saccharolyticus, S.auricularis, S. simulans, S. saprophyticus, S. cohnii, S. xylosus, S.cohnii, S. warneri, S. hyicus, S. caprae, S. gallinarum, S. intermedius,S. hominis.

In a preferred method of the invention said bacterial pathogen is S.aureus or S. epidermidis.

In an alternative preferred method of the invention said bacterialpathogen is selected from the group consisting of: Streptococcuspneumoniae, Pseudomonas aeruginosa or Eschericia coli.

In a further preferred method of the invention said plant derivedproduct is vegetable peptone. Preferably said vegetable peptone includespea flour and/or tryptone soya.

In a further preferred method of the invention said bacterial pathogenis inactivated with chloroform.

In a yet further preferred method of the invention said bacterialpathogen is isolated from said cell culture preparation and freezedried.

According to a further aspect of the invention there is provided aprocess for the production of a vaccine comprising the steps of:

-   -   i) forming a preparation comprising a staphylococcal bacterial        cell;    -   ii) contacting the preparation with an agent that inactivates        the staphylococcal bacterial cell;    -   iii) isolating the inactivated staphylococcal bacterial cell;    -   iv) shearing said preparation to disaggregate the inactivated        bacteria; and optionally    -   v) freeze drying said inactivated staphylococcal bacterial cell.

In a preferred method of the invention said staphylococcal cell isselected from the group consisting of: S. epidermidis, S. aureus, S.hominis, S. haemolyticus, S. warneri, S. capitis, S. saccharolyticus, S.auricularis, S. simulans, S. saprophyticus, S. cohnii, S. xylosus, S.cohnii, S. warneri, S. hyicus, S. caprae, S. gallinarum, S. intermedius,S. hominis.

In a preferred method of the invention said staphylococcal bacterialcell is S. aureus or S. epidermidis.

In a further preferred method of the invention said agent is chloroform.

In a preferred method of the invention shear force is provided by adounce homogenizer.

According to a further aspect of the invention there is provided aStaphylococcus aureus cell as deposited under accession number 13408.

According to a yet further aspect of the invention there is provided abacterial cell culture comprising a Staphylococcus aureus cell asdeposited under accession number 13408.

Throughout the description and claims of this specification, the words“comprise” and “contain” and variations of the words, for example“comprising” and “comprises”, means “including but not limited to”, andis not intended to (and does not) exclude other moieties, additives,components, integers or steps.

Throughout the description and claims of this specification, thesingular encompasses the plural unless the context otherwise requires.In particular, where the indefinite article is used, the specificationis to be understood as contemplating plurality as well as singularity,unless the context requires otherwise.

Features, integers, characteristics, compounds, chemical moieties orgroups described in conjunction with a particular aspect, embodiment orexample of the invention are to be understood to be applicable to anyother aspect, embodiment or example described herein unless incompatibletherewith.

An embodiment of the invention will now be described by example only andwith reference to the following figures:

FIG. 1 illustrates the immune response of placebo subject on westernblotting. Day 1—before vaccination; day 15—after one vaccination; day29—after two vaccinations, day 43 after three vaccinations and day57—after four vaccinations;

FIG. 2 illustrates the immune response of subject given 0.15 mg dose onwestern blotting. Day 1—before vaccination; day 15—after onevaccination; day 29—after two vaccinations; day 43—after threevaccinations; and day 57—after four vaccinations;

FIG. 3 illustrates the immune response of subject given 0.36 mg vaccineon western blotting Day 1—before vaccination; day 15—after onevaccination; day 29—after two vaccinations; day 43—after threevaccinations; and day 57—after four vaccination

FIG. 4 illustrates the immune response of subjects given 0.45 mg vaccineon western blotting. Day 1—before vaccination; day 15—after onevaccination; day 29—after two vaccinations; day 43—after threevaccinations; and day 57—after four vaccinations;

FIG. 5 illustrates the mean change from base line on western blotting ofsera before and after vaccination for vaccinated and placebo subjects.Day 1—before vaccination; day 15—after one vaccination; day 29—after twovaccinations; day 43 after three vaccinations; and day 57—after fourvaccinations;

FIG. 6 illustrates the mean change from baseline against collagenbinding protein of sera before and after vaccination for vaccinated andplacebo subjects;

FIG. 7 illustrates the cross reactivity of anti-sera produced byvaccination of rabbits against a number of unrelated bacteria; M:Molecular weight marker; track 1 E. coli; track 2 Klebsiella edwardii;track 3 Proteus mirabilis; track 4 S. aureus P/DFO 75; track 5 Candidaalbicans; preimmune and hyperimmune serum after 4 inoculations of the S.aureus P/DFO 75 vaccine into a rabbit were used;

FIG. 8 illustrates the production of opsonic antibodies aftervaccination; Subjects 2, 6, 9, and 21 are placebo Day 1 sample is beforevaccination and Day 57 is after 4 vaccinations. Subjects 19, 23, 24, 29,30 are subjects given 0.45 mg vaccine. Subject 3 was given 0.15 mgvaccine;

Table 1 summarises local effects of vaccine administered to subjects;

Table 2 summarises systemic effects of vaccine administered to subjects;

Table 3 summarises immune reactivity of sera from vaccinated and placebosubjects on western blotting;

Table 4 illustrates the presence of antibodies to collagen-bindingprotein in vaccinated and placebo subjects

Table 5 illustrates the longevity of the immune response in vaccinatedsubjects.

MATERIALS AND METHODS Cell Culture and Vaccine Preparation

The bacteria are cultured in tryptone soya both and mixed with 10%glycerol, aliquoted and stored as Master Seed Bank and Working Seed Bankat −70° C. in liquid form and at +4° C. following freeze-drying.Bacteria were prepared by plating out bacteria from one vial of WorkingSeed Bank and growing on tryptone soya agar plates for 16 hours at 37°C. The bacterial growth is then harvested into a small volume oftryptone soya broth which is in turn used to inoculate bigger volumes oftryptone soya broth. The liquid culture is incubated with agitation for16 hours at 37° C. The bacteria are then concentrated by centrifugation.The concentration is adjusted with tryptone soya broth and the cultureshaken with chloroform in a 5:3 culture to chloroform ratio and left tostand for 15-20 minutes at 20° C. to allow phase separation. Thebacterial suspension is top collected, centrifuged at 3-4000 rpm for 15minutes and the pellet resuspended in sterile distilled water orphosphate buffered saline. This is centrifuged again and the pelletresuspended as above.

Alternatively, a staphylococcal strain was isolated in vegetable peptoneagar and subcultured three times in vegetable peptone agar. A liquidculture of bacteria was grown for 16 hours at 37° C. in vegetablepeptone broth and shaken with chloroform and left to stand for 15-20minutes at 20° C. to allow phase separation. The bacterial suspension istop collected, centrifuged and the pellet resuspended in water orphosphate buffered saline, centrifuged and resuspended as above.

Optionally staphylococcal preparations inactivated with chloroform andwashed and resuspended in the relevant medium are immediately frozen andfreeze dried for storage.

EXAMPLES

The vaccine and placebo phosphate buffered saline (PBS) were preparedunder Good Manufacturing Practice (GMP) by the Norwegian Institute ofPublic Health in Oslo, Norway and the double blind placebo controlledPhase I clinical trial was carried out by Simbec Research Limited,Merthyr Tydfil, UK. Testing of sera from volunteers was carried outunder GLP and in house at VRI laboratories and by Professor Jan-IngmarFlock, Karolinska Institute, Stockholm, Sweden.

Forty eight male volunteers between the ages of 18 and 55 were dividedinto 3 groups receiving subcutaneous doses of vaccine SA75 containing0.15 mg, 0.36 mg, or 0.45 mg of protein. In each group 12 volunteersreceived vaccine and 4 received a corresponding placebo phosphatebuffered saline (PBS). A total of four inoculations were administered at2 week intervals. Volunteers remained in the clinical trial unit for 8hours post dosing and were monitored for local (erythema, induration,swelling, haemorrhage, warmth, burning, pruritis and pain) and systemicreactions (malaise, fatigue, flu-like symptoms, feeling of hot/cold,vomiting and headaches) on the day of dosing and on Days 2, 3, and 8post dosing. Blood pressure, ECG, temperature, urinalysis,haematological and biochemical tests were also carried out. Bloodsamples for the purpose of evaluating immune response were taken beforethe first vaccination and two weeks after each of four subsequentvaccinations.

Sera from volunteers before and after vaccination were tested againstwhole chloroform-inactivated homologous organism by ELISA and westernblotting to evaluate immune response to the whole organism. Sera werealso tested by ELISA for the presence of antibodies to collagen bindingprotein, fibrinogen binding protein, fibronectin binding protein andextracellular adherence protein (Professor Jan-Ingmar Flock).

As expected with a whole cell vaccine, transient local reactions wereobserved at the site of injection in vaccinated subjects. There was aclear dose related response and the 0.15 mg and 0.36 mg dosesdemonstrated acceptable mild to moderate levels of local reaction. Adose of 0.45 mg was considered the maximum tolerated dose based upon themore pronounced local reaction. One subject in the 0.45 mg dose grouphad a severe local reaction which led to their withdrawal from the study(Table 1).

There were no significant systemic adverse effects attributable to thevaccine at doses of 0.15 mg and 0.36 mg protein. At the higher dose of0.45 mg there was an increased incidence of pyrexia with a mild increasein temperature reported in 4 subjects. Three subjects receiving the 0.45mg dose experienced general malaise with two classified as mild and oneas severe. No pyrexia or malaise was observed in the placebo, 0.15 and0.36 mg groups. Mild to moderate pain in the extremities was reported inthe 0.45 mg dose group on a number of occasions during the course of thestudy. Severe pain was only reported on a single occasion. Headachewhich was always mild to moderate was reported in all groups includingthe placebo group and increased with increase in dose level (Table 2).

All adverse events were reported over a period of a few hours to severaldays following vaccination and were all transient lasting anything froma few hours to a few days.

On western blotting 75% of the vaccinated subjects demonstrated strongreactivity compared to none of the placebo subjects (p<0.0001) wherestrong reactivity was taken as five or more incidents of increase inpolypeptide reactivity in terms of intensity or number in post comparedto pre-vaccination sera. Weak reactivity was taken as less than fourincreases in polypeptide reactivity in terms of intensity or number inpost compared to pre-vaccination sera; Table and FIGS. 1, 2, 3, 4.

The response was dose related and there was a clear relationship betweenthe immune reactivity of sera and number of vaccinations; FIG. 5.

Antibody levels against collagen binding protein were significantlyincreased in vaccinated volunteers (p0.005) with the number ofresponders increasing with the number of vaccinations; Table 4 and FIG.6. There was no significant increase in antibodies to the other bindingproteins tested.

The chloroform-inactivated S. aureus vaccine designated SA75 was shownto be safe when tested in formal toxicology tests in rabbits andproduced an immune response demonstrated using ELISA and westernblotting in rabbits. This and other additional data then allowed use ofthe vaccine in a double blind placebo controlled trial in humanvolunteers. The SA75 vaccine was shown to be safe and produced an immuneresponse in male volunteers.

There were no clinically significant changes in vital signs, ECGparameters or laboratory safety tests observed during the clinicaltrial. In general there was no difference in systemic response inrelation to the number of vaccinations or dose given.

There was a clear relationship of local reactivity to vaccine dose levelreported, with no discernable difference in local reactions between thedifferent vaccinations at any dose level. There was also a relationshipbetween vaccination dose and the frequency and extent of erythematousreaction but this was not related to the number of vaccinations given.The 0.15 mg and 0.36 mg doses were considered acceptable on safetygrounds.

The immune response measured by ELISA and western blotting demonstrateda clear difference between the frequency of immune response invaccinated subjects compared to those who received placebo.

Cross Reactivity

FIG. 7 illustrates an immunoblot and represents the activity of a rabbitprior to vaccination with the Staphylococcal vaccine (labelled Rabbit 19PI) and following 4 vaccinations (labelled Rabbit 19 after 4vaccinations).

Opsonic Antibodies

FIG. 8 demonstrates the opsonic antibody response in six patients whoreceived vaccination and four patients who received placebo vaccination.There is increase in opsonic antibody level in the vaccinated patientsbut in only one to any significant extent namely patient 2 (placebo). Itis of course perfectly possible that this patient had an intercurrentStaphylococcal infection which would explain this result.

As anticipated there was a highly significant immune response againstthe homologous Staphylococcal strain used to prepare the vaccine in thepenultimate column to the right. There were new immunoreactive bands inthe vaccinated serum against E. coli, Klebsiella and Proteus which arepathogens responsible for a wide variety of human infections includingwound infections and thus there is a reasonable prospect that thevaccine will provide protection against not only Staphylococcal woundinfection but other infections caused by these three and probably otherif not all pathogenic microorganisms. It is also of interest that thevaccinated serum has developed antibodies against Candida albicans—afrequent cause of yeast infections particularly in female subjects—whichis of great interest in as much as Candida albicans is a eukaryoticorganism while Staphylococci and the other organisms tested areprokaryotic organisms. The last observation adds weight to thefeasibility of a Universal vaccine based on our preparative method.

Longevity

Table 5 illustrates that the immune response as adjudged byimmunoblotting which we thought was the most useful indicator of immuneresponse maintains irrespective of vaccine dose for three to six monthsfollowing vaccination. The table does not illustrate an important aspectthat there was an insignificant decline in immune response at 3 and 6months and clearly no subject had returned to the pre vaccination statusby that time. The protocol of the trial regrettably did not allowestimation of antibody levels beyond six months which was perhaps anerror in the formulation of our study.

Subjects who received placebo vaccination and who of course had a baseline level of antibody reactivity against Staphylococcal antigens whichobtains in every subject remained unchanged in three to six monthsindicating an interesting lack of variability in human subjects ingeneral and adds weight to the unequivocal increase in immune reactivityin vaccinated subjects.

1. A composition comprising an inactivated staphylococcal cell whereinsaid cell: i) is a gram positive cocci; ii) expresses at least theenzyme catalase; iii) induces an immune response that producesantibodies that bind at least staphylococcal collagen-binding protein;and iv) is resistant to the antibiotic penicillin.
 2. The compositionaccording to claim 1 wherein said staphylococcal cell expresses theenzymes coagulase and/or Dnase.
 3. The composition according to claim 1wherein said staphylococcal cell induces an immune response thatproduces antibodies that bind collagen binding protein.
 4. Thecomposition according to claim 1, wherein said inactivatedstaphylococcal cell induces an immune response that produces antibodiesthat cross react with methicillin resistant, vancomycin resistant andvancomycin intermediate resistant staphylococcal species.
 5. Thecomposition according to claim 1, wherein said staphylococcal cell issensitive to the antibiotics cloxacillin, erythromycin, tetracycline orgentamicin
 6. The composition according to claim 1, wherein saidstaphylococcal cell is selected from the group consisting of: S.epidermidis, S. aureus, S. hominis, S. haemolyticus, S. warneri, S.capitis, S. saccharolyticus, S. auricularis, S. simulans, S.saprophyticus, S. cohnii, S. xylosus, S. cohnii, S. warneri, S. hyicus,S. caprae, S. gallinarum, S. intermedius, and S. hominis.
 7. Thecomposition according to claim 6, wherein said staphylococcal cell is S.aureus or S. epidermidis.
 8. The composition according to claim 7,wherein said S. aureus or S. epidermidis is antibiotic resistant.
 9. Thecomposition according to claim 8, said S. aureus or S. epidermidis is amethicillin resistant staphylococcal cell (MRSA).
 10. The compositionaccording to claim 8, wherein said S. aureus or S. epidermidis is avancomycin resistant staphylococcal cell (VRSA).
 11. The compositionaccording to claim 1, wherein said staphylococcal cell is Staphylococcusaureus deposited as National Collection of Type Cultures (NCTC)accession number
 13408. 12. The composition according to claim 1,comprising said staphylococcal cell at a protein concentration of notmore than about 1.0 mg or 0.45 mg bacterial protein/ml.
 13. Thecomposition according to claim 1, comprising said staphylococcal cell ata protein concentration of at least 0.0001 mg or 0.1 mg bacterialprotein/ml
 14. The composition according to claim 1, comprising saidstaphylococcal cell at a protein concentration of between 0.0001-1 mgbacterial protein/ml.
 15. The composition according to claim 1,comprising said staphylococcal cell at a protein concentration ofbetween 0.1-0.45 mg bacterial protein/ml.
 16. The composition accordingto claim 1, comprising said staphylococcal cell at between 0.25-0.36 mgbacterial protein/ml.
 17. The composition according to claim 1,comprising said staphylococcal cell at about 0.35 mg bacterialprotein/ml.
 18. The composition according to claim 1, wherein saidvaccine composition comprises an adjuvant and/or excipient.
 19. Thecomposition according to claim 1 further comprising at least oneadditional anti-bacterial agent.
 20. The composition according to claim19, wherein said anti-bacterial agent is a different vaccine and/orimmunogenic agent.
 21. The composition according to claim 1, formulatedfor administration as a nasal spray.
 22. The composition according toclaim 21, provided in an inhaler for delivery of an aerosol.
 23. Aninhaler comprising the composition according to claim
 1. 24-27.(canceled)
 28. A method to vaccinate an animal against a bacterialinfection, comprising administering an effective amount of the vaccinecomposition according to claim 1, thereby vaccinating the animal againstthe bacterial infection.
 29. The method according to claim 28, whereinsaid animal is a human.
 30. The method according to claim 28, whereinsaid bacterial infection is caused by a bacterial pathogen selected fromthe group consisting of: Staphylococcus aureus or Staphylococcusepidermidis; Enterococcus faecalis; Mycobacterium tuberculsis;Streptococcus group B; Streptoccocus pneumoniae; Helicobacter pylori;Neisseria gonorrhoea; Streptococcus group A; Borrelia burgdorferi;Coccidiodes immitis; Histoplasma sapsulatum; Klebsiella edwardii;Neisseria meningitidis type B; Shigella flexneri; Escherichia coli;Haemophilus influenzae, Chalmydia trachomatis, Chlamydia pneumoniae,Chlamydia psittaci, Francisella tularensis, Pseudomonas aeruginos,Bacillus anthracis, Clostridium botulinum, Yersinia pestis, Burkholderiamallei or B pseudomallei.
 31. The method according to claim 28, whereinsaid bacterial pathogen is selected from the group consisting of: S.epidermidis, S. aureus, S. hominis, S. haemolyticus, S. warneri, S.capitis, S. saccharolyticus, S. auricularis, S. simulans, S.saprophyticus, S. cohnii, S. xylosus, S. cohnii, S. warneri, S. hyicus,S. caprae, S. gallinarum, S. intermedius, S. hominis.
 32. The methodaccording to claim 31, wherein said bacterial species is Staphylococcusaureus or Staphylococcus epidermidis.
 33. The method according to claim28, wherein said bacterial infection is caused by an antibioticresistant bacterial species.
 34. The method according to claim 33,wherein said antibiotic resistance bacterial species is a staphylococcalbacterial species.
 35. The method according to claim 33, wherein saidantibiotic resistance staphylococcal species is a methicillin resistantstaphylococcal species (MRSA).
 36. The method according to claim 33,wherein said antibiotic resistance staphylococcal species is avancomycin resistant staphylococcal species (VRSA).
 37. A methodaccording to claim 28, wherein said bacterial infection results in adisease associated with a staphylococcal infection.
 38. A methodaccording to claim 37, wherein said staphylococcal associated disease isselected from the group consisting of: tuberculosis; bacteria-associatedfood poisoning; blood infections; peritonitis; endocarditis;osteomyelitis; sepsis; skin disorders, meningitis; pneumonia; stomachulcers; gonorrhoea; strep throat; streptococcal-associated toxic shock;necrotizing fasciitis; impetigo; histoplasmosis; Lyme disease;gastro-enteritis; dysentery; shigellosis; and arthritis.
 39. A methodaccording to claim 38, wherein said animal is a livestock animal.
 40. Amethod according to claim 39, wherein said livestock animal isvaccinated against bacterial mastitis.
 41. A method according to claim40, wherein said livestock animal is vaccinated against bacterialmastitis caused by gram positive cocci.
 42. A method according to claim41, wherein said gram positive cocci are staphylococcal bacteria, and/orstreptococci bacteria.
 43. A method according to claim 42, wherein saidbacteria are Staphylococcus aureus and/or Streptococcus agalactiae. 44.A method according to claim 35, wherein said livestock animal is acaprine or bovine animal. 45-78. (canceled)
 79. A Staphylococcus aureuscell as deposited under National Collection of Type Cultures (NCTC)accession number
 13408. 80. A bacterial cell culture comprising aStaphylococcus aureus cell as deposited under National Collection ofType Cultures (NCTC) accession number 13408.